Obesity- and alcohol-related diseases are two highly prevalent and important public health problems. Obese individuals who consume alcohol regularly are at high risk of developing fatty liver disease (FLD). Despite the commonality of obesity and alcohol consumption; the mechanisms by which varying amounts of alcohol consumption affect the development and severity of FLD in obese individuals remain poorly understood. The objective of this proposal is to elucidate the mechanisms by which varying amounts (0->60 gm/day) of alcohol consumption affect the development and progression of fatty liver disease in obese subjects. The central hypothesis to be tested is: In obese subjects, alcohol consumption alters the circulating profile of metabolites of intestinal microbial origin which activate circulating macrophages resulting in increased systemic activity of lipoxygenases (LOX) over and above that seen in obesity alone. LOX activation drives the severity of FLD by promoting insulin resistance, oxidative stress, inflammation and apoptosis. Two specific aims are proposed: (Aim 1) Define how alcohol increases the development and severity of FLD in obese subjects by (a) relating alcohol-induced changes in circulating gut microbiome-derived metabolites and eicosanoids to pathways driving fatty liver disease, and changes in intestinal permeability in obese subjects, (b) evaluation of the reversibility of alcohol-induced changes in the systemic profile of gut microbiome-derived metabolites and eicosanoids after successful completion of an alcohol cessation program, and (c) determining the effects of specific intestinal microbiome derived metabolites on LOX expression in vivo and in vitro in macrophages isolated from circulation and (Aim 2): To validate the relevance of LOX activity on the phenotype of FLD in two complementary mouse models of fatty liver disease that will be given isocaloric alcohol feeding via an intragastric route (Tsukamoto method). The animal alcohol feeding will be performed at the NIAAA-funded alcohol research center at the Univ. of Southern California. A systems biology approach will be taken to define the effects of alcohol consumption on systemic exposure to gut microbiome-derived metabolites, impact of such metabolites on LOX expression in circulating macrophages, impact of altered eicosanoid and intestinal microbiome-derived metabolite profile on the hepatic transcriptome and histology and the reversibility of these changes in those who cease to consume alcohol. The effects of specific microbial metabolites on LOX activity will be corroborated by directly testing their effects on specific LOX expression in macrophages isolated from circulation of the study subjects. The role of individual LOX will be determined by a loss of function approach using both pharmacologic and genetic methods to knock down the activity of 5-LOX and 12/15 LOX (aim 2). The investigators are uniquely suited to perform the proposed studies because of their expertise, environment and access to reagents. When these studies are completed, novel information on how alcohol intake modifies systemic LOX activity and the role of such changes in driving liver disease will be obtained.